Gutheil1 1 Division of Pharmaceutical Sciences, University of Missouri-Kansas City, USA;2Department of Pharmacology, University of North Carolina at Chapel Hill, North Carolina, USA;3Dep
Trang 1Overexpression and enzymatic characterization of Neisseria
Miglena E Stefanova1, Joshua Tomberg2, Christopher Davies3, Robert A Nicholas2and William G Gutheil1
1
Division of Pharmaceutical Sciences, University of Missouri-Kansas City, USA;2Department of Pharmacology, University of North Carolina at Chapel Hill, North Carolina, USA;3Department of Biochemistry, Medical University of South Carolina, Charleston, South Carolina, USA
The penicillin-binding proteins (PBPs) are ubiquitous
bac-terial enzymes involved in cell wall biosynthesis,and are the
targets of the b-lactam antibiotics The low molecular mass
Neisseria gonorrhoeaePBP 4 (NG PBP 4) is the fourth PBP
revealed in the gonococcal genome NG PBP 4 was cloned,
overexpressed,purified,and characterized for b-lactam
binding,DD-carboxypeptidase activity,acyl-donor substrate
specificity,transpeptidase activity,inhibition by a number of
active site directed reagents,and pH profile NG PBP 4 was
efficiently acylated by penicillin (30 000M )1Æs)1) Against a
set of five a- and e-substitutedL-Lys-D-Ala-D-Ala substrates,
NG PBP 4 exhibited wide variation in specificity with a
preference for Ne-acylated substrates,suggesting a possible
preference for crosslinked pentapeptide substrates in the cell
wall Substrates with an Ne-Cbz group demonstrated
pro-nounced substrate inhibition NG PBP 4 showed 30-fold
higher activity against the depsipeptide Lac-ester substrate
than against the analogous peptide substrate,an indication that k2(acylation) is rate determining for carboxypeptidase activity No transpeptidase activity was apparent in a model transpeptidase reaction Among a number of active site-directed agents, N-chlorosuccinimide,elastinal,iodoaceta-mide,iodoacetic acid,and phenylglyoxal gave substantial inhibition,and methyl boronic acid gave modest inhibition The pH profile for activity against Ac2-L-Lys-D-Ala-D-Ala (kcat/Km) was bell-shaped,with pKavalues at 6.9 and 10.1 Comparison of the enzymatic properties of NG PBP 4 with otherDD-carboxypeptidases highlights both similarities and differences within these enzymes,and suggests the possibility
of common mechanistic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs Keywords:DD-carboxypeptidase; penicillin-binding protein
Penicillin-binding proteins (PBPs) are ubiquitous bacterial
enzymes that catalyze the last steps in cell wall biosynthesis
(reviewed in [1–5]),and are the targets of the b-lactam
antibiotics In Gram-negative bacteria these enzymes
cata-lyze the reactions shown in Scheme 1 Each bacterial species
has a number of PBPs; for example, Escherichia coli has
eight classically known PBPs,labeled 1A,1B,and 2–7,as
well as several recent additions including PBP 1C [6] and
PBP 6b [7] PBPs have molecular masses of 20–120 kDa and
can be divided into two groups,the low molecular mass
(LMM) PBPs and the high molecular mass (HMM) PBPs
[3] The LMM PBPs have a transpeptidase/hydrolase
domain whereas the HMM PBPs possess an additional
domain N-terminal to the PBP domain,which in some cases catalyze a penicillin-insensitive transglycosylase reaction Different PBPs have different propensities for hydrolysis and/or transpeptidase reactions [5] HMM PBPs are often the lethal targets for b-lactam antibiotics,whereas LMM PBPs are not lethal targets Two LMM PBPs have, however,been found to play important roles in cell division and cell shape; PBP 3 from Streptococcus pneumoniae is required for normal septum formation [8],and PBP 5 from
E coliis essential for normal cell shape [9]
Neisseria gonorrhoeaehas only three PBPs visible on gels when [3H]penicillin G-labeled membranes are analyzed by SDS/PAGE and fluorography (PBPs 1,2,and 3) [10] Analysis of the recently completed genome sequences of
N meningitidis [11] and N gonorrhoeae (GenBank acces-sion number AE004969) revealed a fourth gonococcal PBP, termed PBP 4 (GenBank accession number AF156692) PBPs 1 and 2 are HMM PBPs and are the major antibiotic killing targets for N gonorrhoeae [10] PBP 1 is the gono-coccal homologue of E coli PBP 1A and likely catalyzes both glycan polymerization and transpeptidation during cell elongation [12] PBP 2 is the homologue of E coli PBP 3 and likely functions during cell division [13] PBPs 3 and 4 are LMM PBPs,which are not lethal targets,and their role
in cell wall biosynthesis is unknown In a recently completed study,PBP 3 was found to be an exceptionally active carboxypeptidase,and exhibited high rates of acylation by
Correspondence to W G Gutheil,Division of Pharmaceutical
Sciences,University of Missouri-Kansas City,5005 Rockhill Road,
Kansas City,Missouri 64110.
Fax: +1 816 235 5190,Tel.: +1 816 235 2424,
E-mail: gutheilw@umkc.edu
Abbreviations: Alk-BSA,alkylated BSA; AR,Amplex TM Red; Boc,
tert-butoxycarbonyl; Caps,3-(cyclohexyl amino)-1-propane sulfonic
acid; Cbz,carbobenzyloxy; CPase,carboxypeptidase; EC,Escherichia
coli; HMM,high molecular mass; LMM,low molecular mass; NG,
Neisseria gonorrhoeae; OPD, o-phenylenediamine;
PBP,penicillin-binding protein; KAA, L -Lys- D -Ala- D -Ala; TEA,triethylamine.
(Received 23 September 2003,accepted 21 October 2003)
Trang 2b-lactam antibiotics This study also found that deletion of
PBP 3 and PBP 4 individually had only a slight (P > 0.05,
not statistically significant) effect on the growth and viability
of N gonorrhoeae,whereas deletion of both resulted in a
modest (P < 0.05,statistically significant) decrease in rate
of cell growth Moreover,scanning electron micrographs of
these cells revealed a change in morphology in cells lacking
both PBP 3 and PBP 4,but not in cells lacking only one of
these enzymes,suggesting a role for both enzymes in normal
cell wall biosynthesis [13a]
Characterization of PBPs in terms of enzymatic
proper-ties,structure/function relationships,and catalytic
mechan-ism [14,15] provides useful information for understanding
the role of individual PBPs in bacterial cell wall biosynthesis,
as well as a basis for the development of new inhibitors for
the PBPs [16] that could lead to the development of new
antibacterial agents Enzymatic characterization efforts on
the PBPs have focused on LMM PBPs since in general only
LMM PBPs give detectable enzymatic activity,for reasons
which are as yet unclear There are a number of similarities
between LMM and HMM PBPs in terms of active site
sequence [3] and architecture [14,17,18] Both groups of
enzymes also act on cell wall peptidoglycan as their
substrate,and are reactive with b-lactam antibiotics
Detailed studies of the LMM PBPs are therefore expected
to provide important information on the enzymological
properties of the PBP class of enzymes as a whole,and information on the as yet unexplained functional and catalytic differences between the LMM and HMM PBPs The present study reports the cloning,expression,puri-fication,and enzymological characterization of N gonor-rhoeae(NG) PBP 4 NG PBP 4 was examined for b-lactam binding,DD-carboxypeptidase activity,substrate specificity, transpeptidase activity,sensitivity to general enzyme inhibit-ors,and pH dependence Comparison of the enzymatic properties of NG PBP 4 with otherDD-carboxypeptidases highlights both similarities and differences within these enzymes,and suggests the possibility of common mechan-istic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs
Experimental procedures
General materials and reagents Tris,D-Ala,horseradish peroxidase (Type X; 21 mgÆmL)1
as an ammonium sulfate suspension; 250 UÆmg)1 ),ampi-cillin,FAD,o-phenylenediamine (OPD),diacetyl-L
-Lys-D-Ala-D-Lac (Ac2-KA-D-Lac) andD-lactate dehydrogenase were from Sigma Chemical Co Pig kidneyD-amino acid oxidase (6.0 mgÆmL)1as an ammonium sulfate suspension;
12 UÆmg)1) was from Roche Molecular Biochemicals The PBP/DD-carboxypeptidase substrate diacetyl-L-Lys-D
-Ala-D-Ala (Ac2-KAA) and substituted XY-KAA substrates were synthesized using standard methods of solution phase peptide synthesis [19,20] AmplexTM Red (10-acetyl-3,7-dihydroxyphenoxazine; AR) was from Molecular Probes and the QuantaBluTM substrate solution was from Pierce Chemical Co Protein content was determined by using the Micro Bradford assay (Sigma) according to the manufac-ture’s protocol Alkylated BSA (Alk-BSA) was prepared as described previously [15]
Cloning, expression and protein purification The NG PBP 4 coding sequence was amplified from
N gonorrhoeaestrain FA1090 genomic DNA with primers based on the GenBank sequence (accession number AF156692) The up primer annealed to codons 29–36 inclusive and contained an in-frame BamHI site at its 5¢-end, whereas the down primer annealed to the last seven codons
of the coding sequence and contained an EcoRI site at its 5¢-end The amplified fragment was cloned into a modified pMAL-C2 vector (New England Biolabs),which fused PBP 4 with hexahistidine-tagged maltose-binding protein via a linker sequence containing a cleavage site for Tobacco Etch Virus protease The sequence of the cloned insert was identical to the sequence both from the GenBank entry for
NG PBP 4 and from the completed FA1090 genome (accession numbers AF156692 and AE004969,respectively) The fusion protein was induced with 0.1 mM isopropyl-thio-b-D-galactoside in E coli MC1061 cells and purified from cell lysates on a nickel chelating column The purified fusion protein was cleaved with Tobacco Etch Virus protease,and the digest was repurified on a nickel chelating column PBP 4 did not elute in the flow-through,but instead eluted from the column in 15 mMimidazole,while maltose-binding protein and uncleaved fusion protein eluted
Scheme 1 PBP-catalyzed transpeptidase and carboxypeptidase
reac-tions in E coli.
Trang 3in 150 mMimidazole Purified PBP 4 was estimated to be
> 95% pure by SDS/PAGE,and was stored at)80 C
Determination of the acylation rate of NG PBP 4
by several b-lactam antibiotics
The reaction mechanism for the interaction of PBPs with
peptide and b-lactam antibiotics is:
Eþ S !K0 ES !k2
E S !k3
The constant k2/K¢,which describes the formation of the
covalent acyl-enzyme complex at low (subsaturating)
con-centrations of b-lactams,was determined from time courses
with [14C]penicillin G essentially as described [21] NG PBP 4
(48 lg,1.2 nmol) was diluted into 150 lL binding buffer
(50 mMsodium phosphate pH 7.0,10% glycerol) and mixed
with an equal volume of 100 lM[14C]penicillin G in binding
buffer At timed intervals,20-lL aliquots were removed,
mixedwith5 mL5%trichloroaceticacid(w/v),andincubated
on ice for 15 min The acidified proteins were passed through
#30 glass fiber filters (Schleicher and Schuell) and the filters
were washed twice with 5 mL each of 1% trichloroacetic acid/
33% methanol The filters were then air dried,placed in
scintillation vials with 3 mL Scinti-safe scintillation fluid
(Fisher Scientific),and counted
k2/K¢ constants for ampicillin and ceftriaxone were
determined by the competition method against [14
C]peni-cillin G [21] A fixed concentration of [14C]penicillin G of
0.5 lMand concentrations of the unlabeled antibiotics that
inhibited binding by 50% were incubated with PBP 4 in
sodium phosphate buffer pH 7,at room temperature for
3 min and the level of radioactivity bound to the proteins
was quantified as described above Equation (2) was used to
calculate k2/K¢ constants,where EC0and ECUrepresent the
amount of acyl-enzyme complex formed in the absence and
presence of the unlabeled antibiotic,respectively,and CU
and CLare the concentrations of the unlabeled and labeled
antibiotic,respectively
k2
K0
U
¼ k2
K0
L
ðEC0 ECUÞCL ðECUÞCU
ð2Þ
Enzyme activity assays
D-Ala-D-Ala carboxypeptidase (CPase) activity was
deter-mined by fluorescence-based detection ofD-Ala in
micro-titer plate-based assays as described in detail previously
[15,22] Assays (50 lL) were performed in 100 mM
pyro-phosphate,100 mM NaCl,0.5 mgÆmL)1 Alk-BSA,at
pH 8.5 PBP 4 was diluted in the same buffer,and added
to reactions to start assays Activity against the depsipeptide
(ester) substrate Ac2-KA-D-Lac was determined by
detec-tion ofD-Lac using D-lactate dehydrogenase as described
previously [23],but with assays performed in microtiter
plates and the NADH product measured fluorimetrically
(excitation at 325 nm,emission at 465 nm).D-Lac was used
as a standard Control experiments were performed with
PBP 4 in the absence of substrate,and substrate in
the absence of PBP 4 In the case of the D-Lac based
substrate which has a labile ester bond,a low level of
esterolysis was observed in control experiments minus
NG PBP 4,which was subtracted from experimental values plus NG PBP 4
The linearity of NG PBP 4 catalyzed reactions was verified in a time course experiment NG PBP 4 (105 nM) was added to 10 mM (subsaturating) Ac2-KAA in the standard CPase assay mixture Reactions were stopped at various times by the addition of ampicillin to 50 lgÆmL)1 (135 lM) and the accumulation of the hydrolysis product (D-Ala) was determined fluorimetrically No product (D -Ala) was produced for the zero time point demonstrating that this ampicillin concentration completely blocked PBP 4 activity Additional preliminary experiments further dem-onstrated that the apparent Ki values for ampicillin and penicillin G are in the low nanomolar range (data not shown)
Transpeptidase assays PBPs can catalyze hydrolysis and/or transpeptidase reac-tions (Scheme 1) To determine the ability of NG PBP 4 to catalyze transpeptidase reactions,a model transpeptidase reaction was performed with 10 mMAc2-KAA as the acyl group donor and variable concentrations of glycine as the acyl group acceptor [24,25] NG PBP 4 was added to
175 nMand reactions run for 150 min before stopping by addition of ampicillin This relatively high NG PBP 4 concentration was sufficient to convert 5% of substrate to products,which allowed accurate product determination by HPLC The large amount of D-Ala hydrolysis product produced at this level of turnover was determined using low sensitivity OPD-based microtiter plate assays described previously [23,26] Transpeptidase (Ac2-KAG) and hydro-lysis (Ac2-KA) products were quantified by reverse-phase HPLC on a C18 column (5 lm,0.46· 25 cm) with a water/ acetonitrile gradient The column was equilibrated in 100%
A Gradient: 0–25% B in 15 min [A: 0.1% (v/v) trifluoro-acetic acid in water; C: 0.09% (v/v) trifluorotrifluoro-acetic acid in acetonitrile; B: 30% A/70% C (v/v)]
Substrate specificity
L-Lys-D-Ala-D-Ala (KAA) based substrates with various Na (X) and Ne(Y) substituents were incubated with NG PBP 4
at increasing substrate concentrations (0–50 mM XY-KAA) Data were analyzed by fitting with the appropriate equations by nonlinear regression using BMDP statistical software (SPSS Science)
Effect of inhibitors and reagents Inhibitors and reagents at 1 mM were incubated with the enzyme (21 nM) for 1 h at 25C in an assay mixture containing all components of the CPase assay except for the substrate Ac2-KAA was then added to the assay Reactions were stopped by the addition of developing reagent containing AR and ampicillin as described above The activity of untreated enzyme was taken as 100%
pH dependence and pH stability The effect of pH on NG PBP 4 activity was studied and data analyzed as described in detail previously [15] Activity
Trang 4was assayed in a series of overlapping buffers at 50 mM
buffer,100 mM NaCl,0.5 mgÆmL)1 Alk-BSA pH 3.5–
12.25,with 10 mM Ac2-KAA as the substrate A control
study was performed to determine the effect of pH on
NG PBP 4 stability,also as described previously [15]
Amino acid sequences
The amino acid sequences used in this study were from
the Swiss-Prot database The accession numbers were:
NG PBP 3,O85665; NG PBP 4,Q9XBT7; E coli (EC)
PBP 5,P04287; Streptomyces K15 PBP,P39042; TEM-1
b-lactamase,P00810
Results
b-Lactam binding activity
The gene encoding NG PBP 4 was amplified from N
gon-orrhoeaeFA1090 DNA,expressed in E coli and purified as
described in Experimental procedures To verify that the
purified protein was indeed a PBP,we carried out
penicillin-binding assays with [14C]penicillin G As shown in Table 1,
NG PBP 4 displayed a k2/K¢ acylation rate constant of
30 000M )1Æs)1 with [14C]penicillin G,and an even higher
constant (56 000M )1Æs)1) with ceftriaxone In addition to
these assays,NG PBP 4 activity was found to be completely
inhibited by 140 lM ampicillin used to stop DD-CPase
assays,and an apparent Kiin the low nanomolar range was
observed in additional control experiments (data not
shown)
Substrate specificity
Most LMM PBPs are active as DD-carboxypeptidases
NG PBP 4 was characterized against several D-Ala-D-Ala
based peptide substrates,as summarized in Fig 1 and
Table 2 For data following expected Michaelis–Menten
behavior nonlinear regression with the form of the
Micha-elis–Menten equation shown in Eqn (3) was used to obtain
values and standard errors (SE) for kcatand Km,and the
form of this equation shown in Eqn (4) to obtain the value
and SE for kcat/Km
v=ET¼kcat ½S
v=ET¼ðkcat=KmÞ Km ½S
In the case of Boc-Cbz-KAA and Ac-Cbz-KAA substantial
substrate inhibition was observed (Fig 1) In these two
cases,only the data points from 0 to peak activity were Fig 1 Substrate specificity of NG PBP 4 CPase activity was assayed
as described in the text using 35 n M NG PBP 4,and a reaction time of
90 min D -Ala product was detected with QuantaBlu TM assays j, Boc-Cbz-KAA; r,Boc-Ac-KAA; s,Ac-C-KAA; n,Ac 2 -KAA; h, Boc-H-KAA; d,Ac 2 -KA- D -Lac (A) High activity range showing activity against Ac 2 -KA- D -Lac (B) Middle activity range (C) Low activity range.
Table 1 k 2 /K¢ values for b-lactam antibiotic binding to NG PBP 4.
Standard errors are given in parentheses.
Trang 5included in the statistical analysis using Eqn (4) The kcat/Km
values,which reflect the activity of the enzyme at
subsat-urating (low) substrate concentrations,were determined
using this analysis and are accurate However,the apparent
Kmvalues will be lower than the true Kmvalues,as will the
kcatvalues An effort to fit the full concentration profile to
several substrate inhibition models was attempted,including
noncompetitive and uncompetitive substrate inhibition
models An uncompetitive model [Eqn (5),where Kis is
the substrate inhibition constant] gave the best fit of
experimental data,but the standard errors for kinetic
parameters were high due to overlapping Kisand Kmvalues
The kcat and Km values for these two substrates could
therefore not be resolved,and are not reported
v=ET¼ kcat ½S
Kmþ ½S þ ½S2=Kis
ð5Þ
Values for kcatand Kmfor Boc-Ac-KAA and Boc-H-KAA
also could not be obtained,in these cases due to the lack
of apparent substrate saturation Since the possibility of
substrate inhibition also exists for the other two substrates,
Ac2-KAA and Ac2-KA-D-Lac,the values for kcatand Km
reported in Table 2 are given as the apparent values,and are
the minimum values for these parameters) the true values
could be higher Values for kinetic parameters are also based
on the assumption that all enzyme is catalytically active
NG PBP 4 showed highest activity (kcat/Km) with Na
-acetylated substrates Replacement of the Na-acetyl group
with the bulky Boc group decreased activity Lowest activity
was obtained for Boc-H-KAA,which has a free
(unacyl-ated) Ne NG PBP 4 demonstrated an order of magnitude
higher activity against Ac2-KA-D-Lac (1730M )1Æs)1) than
against the analogous peptide substrate Ac2-KAA
(76M )1Æs)1),with apparent values for Km essentially the
same (60 mMvs 40 mM,respectively)
Transpeptidase activity
No transpeptidase product was observed for NG PBP 4
catalyzed transpeptidation reactions with glycine as the
transpeptidase acceptor and Ac2-KAA as the acyl group
donor A constant amount of accumulated D-Ala with
increasing Gly concentration was also observed (data not
shown) These results demonstrate that NG PBP 4 does not
show transpeptidase activity in a model transpeptidase reaction between Ac2-KAA and Gly
pH optimum and stability The pH dependence of kcat/Km for NG PBP 4 catalyzed hydrolysis of Ac2-KAA is presented in Fig 2A NG PBP 4 gave a bell-shaped pH profile with an optimum in the range 7.5–9.0 Data from TEA buffers demonstrated anomalous behavior and were excluded from the analysis for pKa values The kcat/Kmvs pH data was analyzed to determine the pKavalues as described previously [15] pKavalues were 6.9 (SE¼ 0.1) and 10.1 (0.1) No anomalous effect of buffers other than TEA was observed,except for a slight preference for Caps over carbonate buffers NG PBP 4 was fully stable at pH 5.25–12.25 for 60 min at 25C (Fig 2B)
Fig 2 Effect of pH on activity and stability of NG PBP 4 (A) pH-rate (k cat /K m ) profile for NG PBP 4 hydrolysis of Ac 2 -KAA The NG PBP
4 concentration was 21 n M ,and the reaction time was 75 min D -Ala was detected with AR based assays n,Citrate; s,Pi; r,PPi; e,TEA; d,CO 32–; h,Caps The solid line represents the best fit curve calcu-lated as described in Materials and methods with values of pK 1 ¼ 6.9 (0.1) and pK 2 ¼ 10.1 (0.1) (B) pH stability profile of NG PBP 4 Experimental conditions were as described above Symbols are as in A.
Table 2 Kinetic constants for hydrolysis of D -Ala and D -Lac based
substrates by NG PBP 4 Standard errors are given in parentheses.
H,No substituent; KAA, L -Lys- D -Ala- D -Ala; NS,not available due to
lack of apparent substrate saturation SI,not available due to substrate
inhibition.
Substrate a k cat /K m ( M )1 Æs)1) K m (m M ) b k cat (s)1) b
a
The Na-substituent of Lys is given first,Ne-substituent is given
second b Minimum apparent value as discussed in text.
Trang 6Effect of enzyme inhibitors and reagents
NG PBP 4 was fully inhibited by the oxidizing agent
N-chlorosuccinimide (Table 3) Cysteine modifying
rea-gents iodoacetamide and iodoacetic acid,and the arginine
modifying reagent phenylglyoxal significantly inhibited
enzyme activity (40–50%) Among serine protease
inhibi-tors only elastinal gave substantial inhibition,although
methyl boronic acid showed modest inhibition
Serine-directed organic phosphates were ineffective as inhibitors
None of the tested metal ions or chelators significantly
affected enzyme activity
Discussion
NG PBP 4 was identified from the complete genomic
sequence of N gonorrhoeae Although NG PBP 4 is not
visible when [3H]penicillin G-labeled membranes are
analyzed by SDS/PAGE and fluorography [10],a significant
decrease in growth rate and accompanying morphological
abnormalities occurred only when both PBP 3 (the other
LMM PBP in N gonorrhoeae) and PBP 4 were deleted
[13a],strongly suggesting that PBP 4 plays a role in cell wall synthesis In this study NG PBP 4 was cloned,overex-pressed,purified,and characterized to provide data required for mechanistic and structure–function correlations with other PBPs,and to provide a basis for understanding the possible physiological function of this enzyme
As NG PBP 4 was not observed in [3H]penicillin G labeled membranes [10],it was possible that this protein does not bind b-lactam antibiotics However, b-lactam binding experiments demonstrated that NG PBP 4 does bind [14C]penicillin G and several other b-lactam antibiotics with reasonably high k2/K¢ acylation rate constants (Table 1) Although the k2/K¢ value of [14C]penicillin G with NG PBP 4 is lower than that determined for
NG PBP 3 (198 000M )1Æs)1),it is considerably higher than that of E coli PBP 5,which has a k2/K¢ for [14C]penicillin G
of 390M )1Æs)1[26a] Moreover,NG PBP 4 was completely inhibited by ampicillin at the 140 lMconcentration used to stop carboxypeptidase reactions (data not shown) The lack
of an observable band corresponding to NG PBP 4 in [3H]penicillin G-labeled gonococcal membranes following SDS/PAGE and fluorography therefore cannot be due to a
Table 3 Effect of general enzyme inhibitors and reagents on the carboxypeptidase activity of three LMM PBPs NG PBP 4 data,this paper;
NG PBP 3 data,unpublished observations; EC PBP 5 data,[15].
Inhibitor/reagent
Residual activity (%)
Specificity
Trang 7lack of interaction with penicillin G,and suggests that
N gonorrhoeaegrown in culture expresses only a low level
of this protein Unidentified PBPs with a role in cell wall
morphology have previously been observed in E coli [27]
NG PBP 4 activity against the D-Ala-D-Ala substrate
Ac2-KAA was fairly typical for a PBP with a kcatvalue of
3 s)1, Km value of 3 mM,and kcat/Km of 80M )1Æs)1
(Table 2) NG PBP 4 demonstrated substantial variation
in its activity against a set of XY-L-Lys-D-Ala-D-Ala peptide
substrates and the depsipeptide substrate Ac2-L-Lys-D
-Ala-D-Lac (Table 2,Fig 1) For substituents on the Neof Lys,
Ac- or Cbz-substituted substrates gave highest activity
whereas the absence of either an Ac or Cbz group in
Boc-H-KAA was associated with lowest activity,indicating a
preference of NG PBP 4 for Ne-acylated substrates For
comparison (Table 4),NG PBP 3 demonstrated a similar
preference for Ne-acylated substrates,but in contrast was
more active with bulky substituents on the Na-position,and
EC PBP 5 demonstrated a notable lack of significant
preference for any of these derivatives The observation
that NG PBP 3 and NG PBP 4 show a preference for Ne
-substituted substrates suggests that the preferred substrates
for both LMM neisserial PBPs would be crosslinked
pentapeptides in the cell wall
PBP-catalyzed reactions proceed through an acyl-enzyme
intermediate (Scheme 2) Depsipeptide (peptide-ester)
sub-strates for the PBPs often show a large increase in reaction
rate over the homologous amide substrates [28] Since both
the depsipeptide (Ac2-L-Lys-D-Ala-D-Lac) and amide (Ac2
-L-Lys-D-Ala-D-Ala) substrates proceed through the same
acyl-enzyme intermediate (E-S in Scheme 2), k3will be the
same for both substrates A greater turnover number (kcat)
for a depsipeptide substrate is therefore attributed to a
higher k2,and is an indication that k2(acylation) is the rate
determining step for hydrolysis of the peptide substrate
[28,29] Conversely, if peptide and depsipeptide substrates
show similar turnover numbers then this is evidence that k3
(deacylation) is rate determining NG PBP 4 demonstrated
a much higher kcat(30-fold) against Ac2-L-Lys-D-Ala-D-Lac than against Ac2-L-Lys-D-Ala-D-Ala (Ac2-KAA) (Table 2), evidence that k2 is rate determining for NG PBP 4-catalyzed hydrolysis of Ac2-KAA Classic studies using this approach demonstrated that k2is also rate determining for EC PBP 5-catalyzed hydrolysis of Ac2-KAA [28] In contrast,deacylation (k3) appears rate limiting for both
NG PBP 3 [13a] and S aureus PBP 4 [28,30] catalyzed hydrolysis of amide and ester substrates
A number of active site-directed inhibitors and metal ions were tested for their ability to inhibit NG PBP 4 (Table 3) N-chlorosuccinimide,elastinal,iodoacetamide,iodoacetic acid,and phenylglyoxal gave substantial inhibition,and methyl boronic acid gave modest inhibition As there are no cysteines in NG PBP 4,inhibition by iodoacetamide and iodoacetic acid is attributed to lysine modification Com-parison with inhibitor data from NG PBP 3 and EC PBP 5 (Table 3) demonstrates that all three enzymes were largely unaffected by general inhibitors of serine proteases,which is noteworthy given the role of a serine acyl-enzyme interme-diate in PBP catalysis [28] This result is also consistent with previous observations with the Streptomyces R61 carb-oxypeptidase [31] The only inhibitor effective against all three enzymes was the oxidizing agent N-chlorosuccinimide which inhibited fully NG PBP 4 and NG PBP 3, and to a substantial degree EC PBP 5 (Table 3) Thus,the PBPs appear notably resistant to most active site-directed rea-gents In contrast,the PBPs appear generally sensitive to transition state analogs such as peptide boronic acids [16] and peptide phosphonates [32]
pH dependence is a fundamental enzyme characteristic relevant to physiological,mechanistic,and kinetic under-standing of enzyme catalyzed reactions [33] The physio-logical significance of pH dependence is of particular relevance for the PBPs and the b-lactamases,which are exposed to the extracellular environment pH dependence of
N gonorrhoeaegrowth and cell wall biosynthesis has been investigated ([34] and references therein) N gonorrhoeae grows best at pH 7.2,with a growth window of 5.8–8.4 There is a significant difference in N gonorrhoeae growth environment between the infected female and male,with the vagina having a pH range of 4.2–7.5 and the male urethra having a pH of 6.2–8.4 The pH dependence of NG PBP 4 (pKavalues of 6.9 and 10.1) therefore overlaps the alkaline side of the physiological growth range of N gonorrhoeae in both males and females,with better overlap with male growth conditions The pH profiles for NG PBP 3 and 4 are relatively acidic compared to that of EC PBP 5,which further highlights the contrast between the pH profile of
EC PBP 5 (pKavalues 8.2 and 11.1) and the physiological growth range of E coli (pH 6–8) [15] This is especially notable as EC PBP 5 is required for normal cell shape [9] The pKavalues for NG PBP 4 of 6.9 and 10.1 are almost identical to the pKa values for NG PBP 3 of 6.8 and 9.8 [13a] The pH profile of NG PBP 4,especially the acidic limb,is also similar to the pH profile for the Class A TEM b-lactamase with pKavalues of 5.0–6.2 for the acidic limb and 8.5 for the basic limb [35,36] (Table 5) The conserved W-loop in Class A b-lactamases contains a highly conserved Glu residue (Glu166) which is believed to act as the general
Scheme 2 PBP-catalyzed substrate hydrolysis.
Table 4 Enzyme activities (k cat /K m ) for hydrolysis of D -Ala and D -Lac
based substrates by NG PBP 3, NG PBP 4 and EC PBP 5 NG PBP 4
data,this paper; NG PBP 3 data,[13a]; EC PBP 5 data,[15]
Stan-dard errors are shown in parentheses.
Substrate
Enzyme activity ( M )1 Æs)1)
Ac 2 -KA- D -Lac 1730 (70) 12 300 (800) 700 (10)a
a [23].
Trang 8base in the deacylation step of the catalytic cycle [37,38], and
it is considered responsible for the acidic limb (pKa5–6) in
the pH profile [35] In contrast,EC PBP 5 contains an active
site loop spatially equivalent to theW-loop in b-lactamases
(W-like loop) but which lacks a suitably positioned Glu
residue [14] Although His151 on the W-like loop of
EC PBP 5 aligns with Glu166 in TEM b-lactamase,this
residue does not appear properly oriented to participate in
catalysis [14] and the pKavalues for EC PBP 5
carboxyp-eptidase activity of 8.2 and 11.1 were assigned to Lys47
(SXXK motif) and Lys213 (KTG motif),respectively [15]
NG PBP 4 and NG PBP 3 are both Class C LMM
PBPs,for which no crystal structures are currently
available At the sequence level,Class C LMM PBPs are
most similar to the Class A b-lactamases,followed by
Class A PBPs such as EC PBP 5 and the Streptomyces
K15 enzyme,followed by a relatively remote relationship
to the Class B LMM PBPs such as the Streptomyces R61
enzyme [39] Both gonococcal enzymes are
carboxypeptid-ases,both have the highly conserved active site motifs
common to most members of this class of enzymes,and
both show a readily identifiable W-like loop motif in
sequence alignments (Table 6) However,assigning pKa
values to specific residues in NG PBP 4 and NG PBP 3 is
difficult,especially in the absence of crystal structures By
analogy with TEM b-lactamase and EC PBP 5,the basic
pKaof NG PBP 4 can be reasonably assigned to the KTG
motif lysine (Lys261) (Tables 5 and 6) The acidic limb of
NG PBP 4 at pH 6.9 is however,less easy to assign to
Lys102 and,at first glance,a residue with a more acidic
pKawould seem more appropriate One such candidate is
Glu195,which is present on theW-like loop (Table 6) The
presence of a conserved Gly-Leu/Ile motif at the heart of
the W-like loop suggests that W-like loops may exhibit similar structures within classes A and C of the LMM PBPs Indeed the main chain atoms of those for EC PBP 5 and the Streptomyces K15 transpeptidase can be super-imposed with a root mean square deviation of 0.89 A˚
If so,then Glu195 in NG PBP 4 is unlikely to be suitably positioned to participate directly in catalysis
The absence of other suitable candidates for the acidic
pKa in the active site,at least based on sequence alignment data,focuses attention back to Lys102 as potentially responsible for the acidic pKa Such a low
pKafor a lysine would be unusual but not unprecedented (e.g acetoacetate decarboxylase,pKa 5.9 [40]) A similar argument also applies to NG PBP 3,and suggests Lys61 (SXXK motif) and Lys404 (KTG motif) as most likely responsible for the acidic and basic pKa values,respect-ively (Table 5) Most significantly,this assignment would suggest the same mechanistic function of the two highly conserved Lys residues in NG PBP 4 and NG PBP 3 as
in EC PBP 5 [14,15] and would support a common mechanism of catalysis amongst class A and C LMM PBPs
Note added in proof: A recently reported structural and mutagenesis study of the Streptomyces K15 PBP also implicates the SXXK lysine as the catalytic base during acylation [41]
Acknowledgements Supported by NIH grants GM-60149 (WGG),AI-36901 (RAN),and GM-066861 (CD) We thank Dr Ann E Jerse for providing valuable information and references on N gonorrhoeae growth conditions during review of this manuscript.
Table 6 Sequence alignment of amino acid residues in conserved motifs of NG PBP 3, NG PBP 4, EC PBP 5, TEM b-lactamase and Streptomyces K15 DD -transpeptidase Conserved,mechanistically significant,or potentially mechanistically significant residues are shown in bold.
Protein
Motif
Streptomyces K15 31 RSTG S TT K IMT 41 91 GLMLP S G C DAA 101 138HFDSFD G IGNG 148208 GAIGV K T G SGP 218
b-Lactamase 66 FPMM S TF K VLL 76 125 AAITM S D N TAA 136 162 LDRW E PE L NEA 172 229 WFIAD K S G AGE 239
Table 5 pK a values and residue assignments for TEM b-lactamase, EC PBP 5, NG PBP 3, and NG PBP 4 NA,data not available; AA,amino acid.
pK a
Enzyme
pK a value AA residue pK a value AA residue pK a value AA residue pK a value AA residue
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... the infected female and male,with the vagina having a pH range of 4. 2–7.5 and the male urethra having a pH of 6.2–8 .4 The pH dependence of NG PBP (pKavalues of 6.9 and 10.1) therefore... Nicholas,R.A (1997) Cloning and characterization< /small>of the ponA gene encoding penicillin-binding protein from
Neisseria gonorrhoeae and Neisseria meningitidis... Cloning and characterization of< /small>
PBP 1C,a third member of the multimodular class A
penicillin-binding proteins of Escherichia coli J Biol Chem 2 74, 32031–